In the last several years, appreciation of the importance of mitochondria to cell physiology has risen dramatically. Mitochondria are highly dynamic, undergoing frequent fission and fusion to maintain proper distribution as well as to reduce the effects of oxidative stresses and deleterious mutations in their genome. These attributes are conserved throughout eukaryotes, as demonstrated by evolutionary conservation of the machinery for mitochondrial fission and fusion. The fission machinery is currently thought to consist of a cytosolic dynamin-related GTPase (Drp1 in mammals, Dnm1 in yeast) and mitochondrial protein receptors. However, many aspects of the fission process are unclear. First, the identity of the mitochondrial Drp1 receptor is not clear in mammals, with several proteins (hFis1, Mff, MiD49/51, and GDAP1) being proposed. In addition, it is uncertain whether Drp1-mediated constriction is capable of full mitochondrial fission. Finally, mitochondrial fission appears to be initiated by contact with endoplasmic reticulum (ER), which alone is able to affect a Drp1-independent constriction. We propose a novel mechanism to resolve this issue, with interactions between the ER-mediated actin polymerization driving a primary mitochondrial constriction, which is necessary for a secondary Drp1-based constriction. The ER-bound formin protein INF2 mediates actin polymerization. Our preliminary results provide evidence supporting this mechanism. Our aims utilize cutting-edge techniques (super resolution microscopy, proteomics) to elucidate the macromolecular interactions necessary for ER-mediated mitochondrial fission. Aim 1 uses live-cell confocal and super resolution PALM to track INF2, actin, and Drp1 dynamics during fission at an unprecedented level. Aim 2 uses electron microscopy and super resolution STORM to examine structural features of the fission process and the spatial relationships between the protein components with at least 20 nm resolution. Aim 3 uses proteomics to identify the INF2 receptor on mitochondria, which we postulate is one of the currently hypothesized Drp1 receptors. Proteins identified in Aim 3 will be incorporated into Aims 1 and 2. While we test our mechanistic model, we remain open to many other mechanistic possibilities and our aims are designed to distinguish between these possibilities.